In the state of the art, various methods have been developed to break the diffraction limit in luminescence microscopy. For the purpose of examining biological specimens, in luminescence microscopy, particular dyes (so-called phosphors or fluorophores) are used for specific marking of samples, e.g. cell parts. The sample is illuminated with illumination radiation, which acts as excitation radiation, and the luminescent radiation thus excited is detected by means of suitable detectors. Usually, for this purpose, a dichroic beam splitter is provided in the microscope, in combination with block filters, which separate off the luminescent radiation from the excitation radiation and enable separate observation. This procedure makes it possible to represent individual, differently coloured cell parts in the microscope. Naturally, several parts of a specimen can also be dyed at the same time with differing dyes that attach specifically to differing structures of the specimen. This method is referred to as multiple luminescence. It is also possible to measure samples that are luminescent per se, i.e. without the addition of dye.
Luminescence is understood here, as is common generally, as a generic term for phosphorescence and fluorescence, i.e. it includes both processes. Insofar as reference is made here to fluorescence, that is to be understood as pars pro toto and non-limiting.
A method that achieves a resolution beyond the diffraction limit is known from WO 2006127692 or DE 102006021317 A1. This method, known by the abbreviation PALM (Photo Activated Light Microscopy), uses a marking substance that can be activated by means of optical radiation. It is only in the activated state that the marking substance can then be excited to emit particular fluorescent radiation. Non-activated molecules of the marking substance emit no fluorescent radiation, or at least no appreciable fluorescent radiation, even after introduction of excitation radiation. The activation radiation thus brings the marking substance into a state in which it can be excited to fluorescence. Other types of activation, e.g. of a thermal nature, are also possible. The general term switching signal is therefore used. In the PALM method, then, the switching signal is applied such that at least a certain proportion of the activated marking molecules are at such a distance from adjacent activated molecules that, measured at the optical resolution of the microscopy, they are separate or subsequently separable. The activated molecules are thus at least largely isolated. After recording of the luminescent radiation, the centre of the radiation distribution attributable to resolution limitation is then ascertained for these isolated molecules and, from this, the position of the molecules is determined by computation, with a greater accuracy than is actually afforded by the optical imaging. This increased resolution, through computational centroid determination of the diffraction distribution, is also referred to in the English-language specialist literature as “superresolution”. It requires that, in the sample, at least some of the activated marking molecules be distinguishable, i.e. isolated, with the optical resolution with which the luminescent radiation is detected. For such molecules, specification of the location can then be achieved with increased resolution.
For the purpose of isolating individual marking molecules, the PALM method makes use of the fact that the probability with which a marking module is activated after receiving the switching signal of given intensity, e.g. one photon of the activation radiation, is the same for all molecules. Concerning the intensity of the switching signal, and therefore the number of photons, incident on a unit of area of the sample, it can thus be ensured that the probability of activating marking molecules present in a given surface region of the sample is so low that there are sufficient regions in which only distinguishable marking molecules emit fluorescent radiation within the optical resolution. Through appropriate selection of the intensity, e.g. the photon density, of the switching signal, the result achieved is that, insofar as possible, only marking molecules that are isolated, in terms of the optical resolution, are activated and subsequently emit fluorescent radiation. The determination of location is effected by means of high-sensitivity cameras in wide-field operation with an accuracy down to the nanometer range, if sufficient photons of the isolated marking molecules can be detected. The quasi-punctiform light source, which is constituted by a luminescent marking molecule, is imaged to several camera pixels by the point spread function of the microscope, and the exact position of the luminescent marking molecule in the sample plane assigned to the camera plane can be determined, for example, by fitting the known point spread function (e.g. by means of a Gaussian fit) or centroid determination, etc. Localization accuracies of between 5 and 30 nm are thereby achieved. For localization, various image evaluation methods are known. For this, reference is made, by way of example, to DE 102008009216 A1. For the purpose of imaging the entire sample, the isolation of the marking molecules of the subset by introduction of the activation radiation, subsequent excitation and fluorescent radiation imaging is repeated until, insofar as possible, all marking molecules have been included once in a subset and isolated within the resolution of the imaging operation.
The PALM method has the advantage that a high spatial resolution is not required, either for the activation or for the excitation. Instead, both the activation and the excitation can be effected in wide-field illumination. Individual variants of the PALM method differ principally in the choice of fluorophores and in the type of optical switching process. In the case of molecules that are activated by a separate activation radiation such that they can be excited, the isolation can be effected through suitable application of the activation radiation. Also known, however, are approaches in which, for example, selective bleaching of the sample is used for isolation.
However, this localization accuracy is achieved only laterally, i.e. in a plane assigned to the image plane of the camera. In this respect, the methods are thus limited to a two-dimensional sample analysis. Approaches for localizing luminescent marking molecules in the third spatial direction, which, in relation to the imaging of the sample, is the depth direction, are likewise known from the state of the art.
The publication Huang et al., Science 319, page 810, 2008, proposes introducing into the imaging beam path a low-power cylindrical lens, which results in an astigmatic point spread function. Accordingly, the image of the molecule on the camera is distorted elliptically as soon as the molecule is located above or below the point of symmetry of the point spread function. The information concerning the depth position of the luminescent marking molecule can be obtained from the orientation and the strength of the distortion. A disadvantage of this method is that the local environment and the orientation of a molecular dipole can also result in a distortion of the image of the luminescent marking molecule that has nothing to do with the depth position. An incorrect depth value is then assigned to such luminescent marking molecules, according to their orientation.
The publication Pavani et al., PNAS 106, page 2995, 2009, proposes modifying the point spread function by a spatial phase modulator in the imaging operation to a double helix structure. The point images of individual luminescent marking molecules then become double spots, their depth position being coded in the angular orientation of the common axis of the double spot.
The publication by Shtengel et al., PNAS 106, page 3125, 2009, proposes causing the photons emitted by the luminescent marking molecules to be self-interfering. Used for this purpose are two objective lenses, mounted in 4π configuration, which simultaneously observe the luminescent marking molecules. The partial beam paths obtained in such a manner are brought to a state of interference by means of a special three-way beam splitter. Each of the three-point images received as a result is detected by a camera. The intensity ratios of the three-point images provide an indication of the depth position.
The publications Toprak et al., Nanolet. 7, pages 3285-3290, 2007, and Juette et al., Nature Methods 5, page 527, 2008, describe an approach in which a 50/50 beam splitter is built into the imaging beam path, which beam splitter splits the image of the sample into two sub-images. These two images are detected independently. In addition, in one of the partial beam paths obtained thereby, an optical path-length difference is introduced in such a manner that two object planes, lying apart by approximately half or the entirety of the minimum optical resolution (for example 700 nm) in the z direction, i.e. depth direction, are produced from the two partial beam paths. The depth position of marking molecules that lie between these two planes is then obtained through subtraction of the two sub-images of the same marking molecule, or through corresponding fitting of a three-dimensional point spread function. This method requires two high-sensitivity cameras, or two images have to be arranged next to each other on the receiving region of a high-sensitivity camera, which naturally results in a limitation of the image field. For both options, moreover, a precise adjustment of the beam paths and calibration measurements are essential, in order to achieve a superimposition of the two sub-images that has sub-pixel precision. Furthermore, the two sub-images of a marking molecule generally differ in shape, since the lateral extent of the point spread function of an imaging system changes depending on the position of the object plane observed.